Analytical Post-Embedding Immunogold-Electron Microscopy with Direct Gold-Labelled Monoclonal Primary Antibodies against RIBEYE A- and B-Domain Suggests a Refined Model of Synaptic Ribbon Assembly.

Synaptic ribbons are the eponymous specializations of continuously active ribbon synapses. They are primarily composed of the RIBEYE protein that consists of a unique amino-terminal A-domain and carboxy-terminal B-domain that is largely identical to the ubiquitously expressed transcriptional regulator protein CtBP2. Both RIBEYE A-domain and RIBEYE B-domain are essential for the assembly of the synaptic ribbon, as shown by previous analyses of RIBEYE knockout and knockin mice and related investigations. How exactly the synaptic ribbon is assembled from RIBEYE subunits is not yet clear. To achieve further insights into the architecture of the synaptic ribbon, we performed analytical post-embedding immunogold-electron microscopy with direct gold-labelled primary antibodies against RIBEYE A-domain and RIBEYE B-domain for improved ultrastructural resolution. With direct gold-labelled monoclonal antibodies against RIBEYE A-domain and RIBEYE B-domain, we found that both domains show a very similar localization within the synaptic ribbon of mouse photoreceptor synapses, with no obvious differential gradient between the centre and surface of the synaptic ribbon. These data favour a model of the architecture of the synaptic ribbon in which the RIBEYE A-domain and RIBEYE B-domain are located similar distances from the midline of the synaptic ribbon.

Investigating Retinal Circuits and Molecular Localization by Pre-Embedding Immunoelectron Microscopy.

The retina comprises numerous cells forming diverse neuronal circuits, which constitute the first stage of the visual pathway. Each circuit is characterized by unique features and distinct neurotransmitters, determining its role and functional significance. Given the intricate cell types within its structure, the complexity of neuronal circuits in the retina poses challenges for exploration. To better investigate retinal circuits and cross-talk, such as the link between cone and rod pathways, and precise molecular localization (neurotransmitters or neuropeptides), such as the presence of substance P-like immunoreactivity in the mouse retina, we employed a pre-embedding immunoelectron microscopy (immuno-EM) method to explore synaptic connections and organization. This approach enables us to pinpoint specific intercellular synaptic connections and precise molecular localization and could play a guiding role in exploring its function. This article describes the protocol, reagents used, and detailed steps, including (1) retina fixation preparation, (2) pre-embedding immunostaining, and (3) post-fixation and embedding.

How to apply the broad toolbox of correlative light and electron microscopy to address a specific biological question.

Correlative light and electron microscopy (CLEM) is an approach that combines the strength of multiple imaging techniques to obtain complementary information about a given specimen. The "toolbox" for CLEM is broad, making it sometimes difficult to choose an appropriate approach for a given biological question. In this chapter, we provide experimental details for three CLEM approaches that can help the interested reader in designing a personalized CLEM strategy for obtaining ultrastructural data by using transmission electron microscopy (TEM). First, we describe chemical fixation of cells grown on a solid support (broadest approach). Second, we apply high-pressure freezing/freeze substitution to describe cellular ultrastructure (cryo-immobilization approach). Third, we give a protocol for a ultrastructural labeling by immuno-electron microscopy (immuno-EM approach). In addition, we also describe how to overlay fluorescence and electron microscopy images, an approach that is applicable to each of the reported different CLEM strategies. Here we provide step-by step descriptions prior to discussing possible technical problems and variations of these three general schemes to suit different models or different biological questions. This chapter is written for electron microscopists that are new to CLEM and unsure how to begin. Therefore, our protocols are meant to provide basic information with further references that should help the reader get started with applying a tailored strategy for a specific CLEM experiment.

Ultrastructural localization of calcium homeostasis modulator 1 in mouse taste buds.

Taste receptor cells are morphologically classified as types II and III. Type II cells form a unique type of synapses referred to as channel synapses where calcium homeostasis modulator 1 (CALHM1) together with CALHM3 forms voltage-gated channels that release the neurotransmitter, adenosine triphosphate (ATP). To validate the proposed structural model of channel synapses, the ultrastructural localization of CALHM1 in type II cells of both fungiform and circumvallate taste buds was examined. A monoclonal antibody against CALHM1 was developed and its localization was evaluated via immunofluorescence and immunoelectron microscopy using the immunogold-silver labeling technique. CALHM1 was detected as puncta using immunofluorescence and along the presynaptic membrane of channel synapses facing atypical mitochondria, which provide ATP, by immunoelectron microscopy. In addition, it was detected along the plasma membrane lined by subsurface cisternae at sites apposed to afferent nerve fibers. Our results support the validity of a previously proposed structural model for channel synapses and provide insights into the function of subsurface cisternae whose function in taste receptor cells is unknown. We also examined the localization of CALHM1 in hybrid synapses of type III cells, which are conventional chemical synapses accompanied by mitochondria similar to atypical mitochondria of channel synapses. CALHM1 was not detected in the six hybrid synapses examined using immunoelectron microscopy. We further performed double immunolabeling for CALHM1 and Bassoon, which is detected as puncta corresponding to conventional vesicular synapses in type III cells. Our observations suggest that at least some, and probably most, hybrid synapses are not accompanied by CALHM1.

Pre- and Postembedding Immunoelectron Microscopy to Analyze the Central Nervous System.

Immunocytochemistry, a method of delineating the subcellular localization of target proteins, was developed from immunohistochemistry. In principle, proteins are labeled using an antigen-antibody reaction. In order to observe under an electron microscope, the reaction product must scatter the electron beam with sufficient contrast while it is necessary to have an amplifying label that can withstand the observation. We have some detailed tips on making electron microscope samples to achieve this objective, and we would be happy to help you.

Localization of Diacylglycerol Kinase ζ in the Kidney of Adult Rats: Its Dominant Expression in Collecting Tubules and its Selective Lack in Proximal Tubules / Localización de Diacilglicerol Quinasa ζ en el Riñón de Ratas Adultas: su Expresión Dominante en Túbulos Colectores y su Carencia Selectiva en Túbulos Proximales

SUMMARY: Diacylglycerol kinase (DGK) exerts balancing the intracellular level between two-second messengers, diacylglycerol and phosphatidic acid, by its phosphorylation activity. DGK ζ is often localized in cell nuclei, suggesting its involvement in the regulation of intranuclear activities, including mitosis and apoptosis. The present immunohistochemical study of rat kidneys first revealed no detection levels of DGK ζ -immunoreactivity in nuclei of most proximal tubule epithelia in contrast to its distinct occurrence in cell nuclei of collecting and distal tubules with the former more dominant. This finding suggests that DGK ζ is a key factor regulating vulnerability to acute kidney injury in various renal tubules: its low expression represents the high vulnerability of proximal tubule cells, and its distinct expression does the resistance of collecting and distal tubule cells. In addition, this isozyme was more or less localized in nuclei of cells forming glomeruli as well as in endothelial nuclei of peritubular capillaries and other intrarenal blood vessels, and epithelial nuclei of glomerular capsules (Bowman's capsules) and renal calyces, including intrarenal interstitial cells.

La diacilglicerol quinasa (DGK) ejerce el equilibrio del nivel intracelular entre dos segundos mensajeros, diacilglicerol y ácido fosfatídico, por su actividad de fosforilación. La DGK ζ a menudo se localiza en los núcleos celulares, lo que sugiere su participación en la regulación de las actividades intranucleares, incluidas la mitosis y la apoptosis. El presente estudio inmunohistoquímico en riñones de rata no reveló niveles de detección de inmunorreactividad de DGK ζ en los núcleos de la mayoría de los epitelios de los túbulos proximales, en contraste a la detección en los núcleos celulares de los túbulos colectores y distales, siendo el primero más dominante. Este hallazgo sugiere que DGK ζ es un factor clave que regula la vulnerabilidad a la lesión renal aguda en varios túbulos renales: su baja expresión representa la alta vulnerabilidad de las células del túbulo proximal, y su expresión distinta hace a la resistencia de las células del túbulo colector y distal. Además, esta isoenzima estaba más o menos localizada en los núcleos de las células que forman los glomérulos, así como en los núcleos endoteliales de los capilares peritubulares y otros vasos sanguíneos intrarrenales, y en los núcleos epiteliales de las cápsulas glomerulares (cápsulas de Bowman) y los cálices renales, incluidas las células intersticiales intrarrenales.

Immunohistochemical and Immunoelectron Microscopical Distribution of MEGF8 in the Mouse Central Nervous System.

Mutations in multiple epidermal growth factor-like domain 8 (MEGF8), a multidomain transmembrane protein encoded by a gene conserved across species, cause Carpenter's syndrome, which is associated with learning disabilities, mental health issues, and left-right patterning abnormalities. MEGF8 interacts with MGRN1, a protein that functions as an E3 ubiquitin ligase and is involved in multiple physiological and pathological processes. However, the mechanism underlying the distribution of MEGF8 in the central nervous system (CNS) and its cellular and subcellular locations remain unknown. This study aimed to map MEGF8 in the mouse CNS using a new antibody. We discovered that MEGF8 was distributed in the majority of neuronal cell somata across most CNS regions. High levels of MEGF8 were expressed in the neuropils of the CNS gray matter. Immunoelectron microscopy showed that MEGF8 was present in the synapses and around the outer mitochondrial membrane. These findings show that MEGF8 is uniformly distributed throughout the mouse CNS, and its distribution indicates that it plays a substantial role in synaptic and mitochondrial functions. To the best of our knowledge, this is the first study to document MEGF8 distribution in the CNS.

Spatiotemporally differential expression of HER (human epithelial growth factor receptor)-2 with sexual dimorphism in submandibular gland of mice

SUMMARY: Considering that the submandibular gland (SMG) of postnatal mice performs active cell proliferation, apoptosis and differentiation which are regulated by proto-oncogene products in cancerous cells, the expression and localization of a proto-oncogene product HER (human epidermal growth factor receptor)-2 was examined in SMG of postnatal mice. In Western blot analysis, the expression for HER-2 was high until pre-puberty, and it decreased from puberty to young adult stages with male SMG more dominant. In immunohistochemistry, the immunoreactivity was positive in acinar and ductal cells of newborn SMG with distinct localization at the intercellular apposition sites. The immunoreactivity in acinar cells progressively decreased to negligible levels by pre-pubertal stage, while it remained positive in most ductal cells throughout the postnatal time-course. The immunoreactivity in cells of terminal tubules and intercalated ducts, both of which have a high potential to produce cells, were seen at levels similar to those of more proximal ducts, while the immunoreactivity in ductal basal cells was significantly high, but the granular convoluted tubule cells were seen at negligible levels in male and at faint levels in female. In immuno-electron microscopy of excretory ducts, the immunoreactivity was dominantly localized on the basal infolding membranes as well as vesicles and vacuoles of various sizes, but rarely in Golgi apparatus and mitochondria. The immunoreactivity without association to any membranous structures were also seen, though not numerous. The relation of expression levels of HER-2 in various portions of normal SMG to those in their cancerous ones is briefly discussed.

RESUMEN: Considerando que la glándula submandibular (GSM) de ratones postnatales realiza la proliferación celular activa, apoptosis y diferenciación que están reguladas por productos protooncogénicos en células cancerosas, la expresión y localización de un producto protooncogénico HER (receptor del factor de crecimiento epidérmico humano) - 2 se examinó en GSM de estos ratones. En el análisis de Western blot, la expresión de HER-2 fue alta hasta la prepubertad, y disminuyó desde la pubertad hasta las etapas de adultos jóvenes con GSM macho más dominante. En inmunohistoquímica, la inmunorreactividad fue positiva en las células acinares y ductales de GSM de recién nacido con una localización distinta en los sitios de aposición intercelular. La inmunorreactividad en las células acinares disminuyó progresivamente a niveles insignificantes en la etapa prepuberal, mientras que permaneció positiva en la mayoría de las células ductales durante el transcurso del tiempo posnatal. La inmunorreactividad en las células de los túbulos terminales y los conductos intercalados, los cuales tienen un alto potencial para producir células, se obser- vó a niveles similares a los de los conductos más proximales, mientras que la inmunorreactividad en las células basales ductales fue significativamente alta, pero en el túbulo contorneado granular las células se observaron en niveles insignificantes en los machos y en niveles débiles en las hembras. En la microscopía inmunoelectrónica de los conductos excretores, la inmunorreactividad se localizó de manera predominante en las membranas de pliegues basales, así como en vesículas y vacuolas de varios tamaños, pero raramente en el aparato de Golgi y en las mitocondrias. También se observó la inmunorreactividad sin asociación a ninguna estructura membranosa, aunque no numerosa. Se discute brevemente la relación de los niveles de expresión de HER-2 en varias porciones de GSM normal con aquellos en sus cancerosos.

Helicobacter pylori Antigens Inducing Early Immune Response in Infants

To identify the Helicobacter pylori antigens operating during early infection in sera from infected infants using proteomics and immunoblot analysis. Two-dimensional (2D) large and small gel electrophoresis was performed using H. pylori strain 51. We performed 2D immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) antibody immunoblotting using small gels on sera collected at the Gyeongsang National University Hospital from 4–11-month-old infants confirmed with H. pylori infection by pre-embedding immunoelectron microscopy. Immunoblot spots appearing to represent early infection markers in infant sera were compared to those of the large 2D gel for H. pylori strain 51. Corresponding spots were analyzed by matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS). The peptide fingerprints obtained were searched in the National Center for Biotechnology Information (NCBI) database. Eight infant patients were confirmed with H. pylori infection based on urease tests, histopathologic examinations, and pre-embedding immunoelectron microscopy. One infant showed a 2D IgM immunoblot pattern that seemed to represent early infection. Immunoblot spots were compared with those from whole-cell extracts of H. pylori strain 51 and 18 spots were excised, digested in gel, and analyzed by MALDI-TOF-MS. Of the 10 peptide fingerprints obtained, the H. pylori proteins flagellin A (FlaA), urease β subunit (UreB), pyruvate ferredoxin oxidoreductase (POR), and translation elongation factor Ts (EF-Ts) were identified and appeared to be active during the early infection periods. These results might aid identification of serological markers for the serodiagnosis of early H. pylori infection in infants.

Identification in immuno-electron microscopy of solitary multi-polar peripheral neurons of adult Opisthorchis viverrini by antibody against rat diacylglycerol kinase / Identificación por microscopía imuno-electrónica de las neuronas periféricas multipolares solitarias del Opisthorchis viverrini adulto mediante anticuerpo contra la diacilglicerol kinasa de ratas

By utilizing the antibody for rat DGKz a substantial number of immunopositive cells were found in the OV (Opisthorchis viverrini). The immunopositive cells appeared solitarily and they were distributed rather symmetrically to the longitudinal axis of the OV. Some of them were located in close proximity to internal organs such as uterus, ovary, testes, vitelline glands and guts. The immunostained cells extended tapering processes horizontally or obliquely to the OV longitudinal axis. In immuno-electron microscopy, the immunopositive cells were characterized by intensely immunostained mitochondria and weakly immunostained cytoplasm and immunonegative chromatin-poor nucleus. Vacuoles of various sizes without the immunoreactivity were also contained in the cells. Thin cellular processes without the immunoreactivity were found to enclose thinly the entire surfaces of the immunostained cells and processes, and they were in continuity with the interstitial partition-like processes which contained nuclei and aggregation of microfibrils at some distance from the cytoplasmic envelopes. The present finding suggests the possibility that the immunostained cells were peripheral neurons enveloped by peripheral glia and that the glia are of mesenchymal origin because of their cytoplasmic continuity to the interstitial partition-like processes. The motor or sensory nature of the neurons remains to be elucidated.

Mediante el uso del anticuerpos DGK para rata se determinó un número considerable de células inmunopositivas en el Opisthorchis viverrini (OV). Las células inmunopositivas aparecían solitarias y se distribuían simétricamente al eje longitudinal de la OV. Algunas estaban ubicadas en las proximidades de los órganos internos como el útero, ovarios, testículos, glándulas vitelinas e intestino. Las células inmunoteñidas extendían sus procesos horizontalmente u oblicuamente al eje longitudinal de la OV. Por microscopía inmunoelectrónica, las células inmunopositivas se caracterizaron por presentar mitocondrias intensamente teñidas, citoplasma con tinción débil e inmunonegatividad en núcleos pobres en cromatina. También se observó en las células, vacuolas de diversos tamaños sin inmunorreactividad. Se encontraron procesos celulares sin inmunorreactividad para cerrar finamente todas las superficies de las células y procesos, y se continuaron con los procesos de partición intersticiales que contenían núcleos y agregación de microfibrillas a cierta distancia de las envolturas citoplásmicas. El presente hallazgo sugiere la posibilidad de que las células inmunoteñidas son neuronas periféricas envueltas por glia periférica y que la glía presenta origen mesenquimal debido a su continuidad citoplasmática con los procesos de partición intersticiales. La naturaleza motora o sensorial de las neuronas aún no se ha dilucidado.

GLCCI1 is a novel component associated with the PI3K signaling pathway in podocyte foot processes

Podocyte foot processes are interdigitated to form the slit diaphragm and are crucial for the glomerular filtration barrier. Glucocorticoid-induced transcript 1 (GLCCI1) is transcriptionally regulated, but its signaling pathway in podocytes is unknown. The main objective of this study was to investigate the regulation of podocyte foot process proteins and to investigate the role of GLCCI1 in the phosphoinositide 3-kinase (PI3K) pathway using high glucose-induced podocytes and streptozotocin-induced diabetic rats. In podocytes and rat kidneys, GLCCI1 was found to be highly specific for the glomerulus and podocyte foot processes similar to other podocyte-specific proteins (nephrin, podocin, synatopodin and podocalyxin) based on reverse transcription-PCR, western blotting, immunofluorescence and immunoelectron microscopy analyses. In addition, the decrease in the GLCCI1 expression level under hyperglycemic conditions was restored by treatment with a PI3K inhibitor (wortmannin). Immunofluorescence analysis confirmed that GLCCI1 colocalized with nephrin and synaptopodin both in vivo and in vitro. Finally, immunoelectron microscopy data from streptozotocin-induced diabetic rats showed that GLCCI1 also localized in podocyte foot processes. Hence, GLCCI1 is a component of podocyte foot processes, and its expression appears to be regulated via the PI3K pathway.

Hydration status affects osteopontin expression in the rat kidney

Osteopontin (OPN) is a secretory protein that plays an important role in urinary stone formation. Hydration status is associated with the development of urolithiasis. This study was conducted to examine the effects of dehydration and hydration on OPN expression in the rat kidney. Animals were divided into three groups, control, dehydrated, and hydrated. Kidney tissues were processed for light and electron microscope immunocytochemistry, in situ hybridization, and immunoblot analysis. Dehydration induced a significant increase in OPN protein expression, whereas increased fluid intake induced a decrease in protein expression. Under control conditions, OPN protein and mRNA expression were only detected in the descending thin limb (DTL). Dehydration induced increased expression in the DTL and the development of detectable expression in the thick ascending limb (TAL). In contrast, OPN expression levels declined to less than the controls in the DTL after hydration, while no expression of either protein or mRNA was detectable in the TAL. Immunoelectron microscopy demonstrated that hydration status altered tubular ultrastructure and intracellular OPN expression in the Golgi apparatus and secretory cytoplasmic vesicles. These data confirm that changes in oral fluid intake can regulate renal tubular epithelial cell OPN expression.

Propuesta de reformas a los prestadores públicos de servicios médicos en Chile: “fortaleciendo la opción pública” / A reform proposal to strengthen public health care

Currently, there is no discussion on the need to improve and strengthen the institutional health care modality of FONASA (MAI), the health care system used by the public services net and by most of the population, despite the widely known and long lasting problems such as waiting lists, hospital debt with suppliers, lack of specialists and increasing services purchase transference to the private sector, etc. In a dichotomous sectorial context, such as the one of health’s social security in Chile (the state on one side and the market on the other), points of view are polarized and stances tend to seek refuge within themselves. As a consequence, to protect the public solution is commonly associated with protecting the “status quo”, creating an environment that is reluctant to change. The author proposes a solution based on three basic core ideas, which, if proven effective, can strengthen each other if combined properly. These are: network financing management, governance of health care services in MAI and investments and human resources in networked self-managed institutions. The proposal of these core ideas was done introducing a reality testing that minimizes the politic complexity of their implementation.

Diagnosis, classification and treatment of gestational trophoblastic neoplasia / Diagnóstico, classificação e tratamento da neoplasia trofoblástica gestacional

Gestational trophoblastic neoplasia (GTN) is the term to describe a set of malignant placental diseases, including invasive mole, choriocarcinoma, placental site trophoblastic tumor and epithelioid trophoblastic tumor. Both invasive mole and choriocarcinoma respond well to chemotherapy, and cure rates are greater than 90%. Since the advent of chemotherapy, low-risk GTN has been treated with a single agent, usually methotrexate or actinomycin D. Cases of high-risk GTN, however, should be treated with multiagent chemotherapy, and the regimen usually selected is EMA-CO, which combines etoposide, methotrexate, actinomycin D, cyclophosphamide and vincristine. This study reviews the literature about GTN to discuss current knowledge about its diagnosis and treatment.

Neoplasia trofoblástica gestacional (NTG) é o termo que descreve o conjunto de anomalias malignas da placenta, incluindo a mola invasora, coriocarcinoma, tumor trofoblástico do sítio placentário e tumor trofoblástico epitelióide. Ambos a mola invasora e o coriocarcinoma respondem bem à quimioterapia, com taxas de cura superiores a 90%. Desde o advento da quimioterapia, NTG de baixo risco tem sido tratada com monoquimioterapia, pelo geral methotrexate ou actinomicina-D. Casos de NTG de alto risco, contudo, devem ser tratados com poliquimioterapia, e o regime usualmente escolhido é o EMA-CO que combina etoposide, methotrexate, actinomicina-D, ciclofosfamida e vincristina. Esse estudo revê a literatura sobre NTG a fim de discutir os conhecimentos atuais sobre seu diagnóstico e tratamento.

Characterization of fibrillous inclusion body (FIB) of human adenovirus type 41 (Ad41) using immunoelectron microscopy / 病毒学报

To investigate the components of fibrillous inclusion body (FIB), which was formed in packaging cells during the replication of human adenovirus type 41 (Ad41), Ad41 long fiber knob (LFK) and short fiber knob (SFK) proteins were expressed in prokaryote respectively and then used to immunize BALI mice for preparation of anti-LFK serum and anti-SFK sera. The activity and specificity of anti-LFK and an ti-SFK sera were confirmed with Western blot, indirect immunofluorescence assay (IFA) and immunonegative staining, suggesting these sera could be applied in immuno-colloidal gold labelling electron microscopy (EM). 293TE cells were infected with wild-type Ad41. Ultrathin sections of infected cells were made, and labelled with immuno-colloidal gold technique using anti-Ad41 sera, anti-LFK sera, anti-SFK sera, or anti-fiber monoclonal antibody 4D2, respectively. The labelled sections were observed under EM, and the results demonstrated that both Ad41 long fiber protein and short fiber protein were included FIB.

Immunoelectron microscopy study of superficial skin nerves in drug-induced acute urticaria / Estudo de microscopia imunoeletrônica dos nervos superficiais da pele na urticária aguda induzida por medicamentos

BACKGROUND: Few studies have evaluated the ultrastructure of the superficial skin nerves in urticaria. OBJECTIVE: The objective of this study was to describe findings in superficial skin nerves in cases of drug-induced acute urticaria. METHODS: Seven patients with drug-induced acute urticaria were included in the study. Skin biopsies were obtained from the urticarial lesion and from the apparently normal skin. The 14 fragments collected were processed for immunogold electron microscopy using single stains for antitryptase and anti-FXIIIa antibodies, as well as double immunogold labeling for both. RESULTS: Some sections showed mast cells in the process of degranulation. Following double immunogold staining, 10 nm (FXIIIa) and 15 nm (Tryptase) gold particles were found together throughout the granules in mast cells, indicating that tryptase and FXIIIa are located inside each one of the granules of these cells. Interestingly, we found strong evidence of the presence of tryptase and factor XIIIa in the superficial skin nerves of these patients, both in cases of urticarial lesions (wheals) and in the apparently normal skin. CONCLUSIONS: Tryptase and FXIIIa are present in the superficial nerves of the skin in drug-induced acute urticaria. This is the first report of tryptase and FXIIIa expression in the superficial skin nerves of patients with urticaria. Tryptase may be participating in neural activation in these patients, while FXIIIa may be present in the nerves to guarantee the functional integrity of structures.

FUNDAMENTOS: Poucos autores têm estudado a ultraestrutura dos nervos superficiais na urticária. OBJETIVO: Descrever os achados nos nervos cutâneos superficiais em casos de urticária aguda induzida por medicamentos. MÉTODOS: Sete pacientes com urticária aguda induzida por medicamentos foram incluídos no estudo. Foram obtidas biopsias da pele da lesão urticariforme e da pele aparentemente normal. Os 14 fragmentos coletados foram processados usando imunomarcação com ouro para anticorpos anti-triptase e anti-FXIIIa separadamente, além da dupla imunomarcação com ambos anticorpos. A seguir as amostras foram submetidas à análise por microscopia imunoeletrônica. RESULTADOS: Alguns cortes demonstraram mastócitos em processo de degranulação. Após a imunomarcação dupla, partículas de ouro de 10 nm (FXIIIa) e partículas de ouro de 15 nm (Triptase) apresentavam-se juntas em grânulos de mastócitos indicando que a triptase e o FXIIIa se localizam dentro de cada um dos grânulos dessas células. Curiosamente, foi encontrada uma forte evidência da presença da triptase e do fator XIIIa nos nervos superficiais dos pacientes avaliados, tanto em lesões urticadas, como na pele aparentemente normal. CONCLUSÕES: A triptase e o FXIIIa estão presentes nos nervos superficiais da pele na urticária aguda medicamentosa. Este é o primeiro relato da expressão de triptase e de FXIIIa nos nervos superficiais na urticária. A triptase poderia estar participando da ativação neural nos pacientes estudados. O FXIIIa poderia estar presente nos nervos, com a finalidade de manter a integridade funcional dessas estruturas.

Correlative microscopy of Purkinje cells

The Purkinje cell and their synaptic contacts have been described using (1) light microsocopy, (2) transmission and scanning electron microscopy, and freeze etching technique, (3) conventional and field emission scanning electron microscopy and cryofracture methods, (4) confocal laser scanning microscopy using intravital stain FM64, and (5) immunocytochemical techniques for Synapsin-I, PSD9-5, GluR1 subunit of AMPA receptors, N-cadherin, and CamKII alpha. The outer surface and inner content of plasma membrane, cell organelles, cytoskeleton, nucleus, dendritic and axonal processes have been exposed and analyzed in a three-dimensional view. The intramembrane morphology, in bi- and three-dimensional views, and immunocytochemical labeling of synaptic contacts with parallel and climbing fibers, basket and stellate cell axons have been characterized. Freeze etching technique, field emission scanning microscopy and cryofracture methods, and GluR1 immunohistochemistry showed the morphology and localization of postsynaptic receptors. Purkinje cell shows N-cadherin and CamKII alpha immunoreactivity. The correlative microscopy approach provides a deeper understanding of structure and function of the Purkinje cell, a new three-dimensional outer and inner vision, a more detailed study of afferent and intrinsic synaptic junctions, and of intracortical circuits.

Correlative microscopy of Purkinje cells

The Purkinje cell and their synaptic contacts have been described using (1) light microsocopy, (2) transmission and scanning electron microscopy, and freeze etching technique, (3) conventional and field emission scanning electron microscopy and cryofracture methods, (4) confocal laser scanning microscopy using intravital stain FM64, and (5) immunocytochemical techniques for Synapsin-I, PSD9-5, GluR1 subunit of AMPA receptors, N-cadherin, and CamKII alpha. The outer surface and inner content of plasma membrane, cell organelles, cytoskeleton, nucleus, dendritic and axonal processes have been exposed and analyzed in a three-dimensional view. The intramembrane morphology, in bi- and three-dimensional views, and immunocytochemical labeling of synaptic contacts with parallel and climbing fibers, basket and stellate cell axons have been characterized. Freeze etching technique, field emission scanning microscopy and cryofracture methods, and GluR1 immunohistochemistry showed the morphology and localization of postsynaptic receptors. Purkinje cell shows N-cadherin and CamKII alpha immunoreactivity. The correlative microscopy approach provides a deeper understanding of structure and function of the Purkinje cell, a new three-dimensional outer and inner vision, a more detailed study of afferent and intrinsic synaptic junctions, and of intracortical circuits.(AU)

An immunohistochemical study on the initial development of 155 kd neurofascin in rat sciatic nerve / 대한수의학회지

Neurofascin, one of the members of L1CAM, has been known to have some important roles during the development of nerve fibers. In order to investigate the role of neurofascin associated with the development of nerve fibers in the rat sciatic nerve, the initial development of NF155 in the paranode was studied with immuno-fluorescence and immuno-electron microscopy. The result of the present study showed NF155 was not detected in the fetal sciatic nerve and began to reveal at the postnatal day 0 (P0) and dramatically increased by time lapse until postnatal day 7 (P7). NF155 was prominently localized in the axolemma of paranode and not detected in the central region of node of Ranvier. According to the present study, NF155 is likely to have some relationships with the formation of paranode and myelin sheath.

Ribbon Synapses Formed at the Axon of the Calbindin-Immunoreactive ON Cone Bipolar Cell in OFF Sublamina of the Inner Plexiform Layer in the Rabbit Retina / 체질인류학회지

Some retinal neurons, including intrinsically photosensitive retinal ganglion cells have their dendrites stratified in sublamina a of the inner plexiform (IPL), the OFF sublayer, but paradoxically show light-driven ON electrophysiological responses. In order to understand the mechanism on this paradoxical response, by using immunoelectron microscopy with a specific antibody against calbindin, we examined the synaptic connections of the calbindin-immunoreactive ON cone bipolar cell of the rabbit retina, which is thought to make the ribbon synapse in sublamina a of the IPL. The ribbon synapses in sublamina a by calbindin-immunoreactive ON cone bipolar cells were mainly found at the border between the inner nuclear layer and the IPL. Interestingly, the output targets at these ribbon synapses turned out as monads, and multiple synaptic ribbons were engaged in each synapse. These findings were different from those at the conventional ribbon synapse formed by calbindin-immunoreactive ON cone bipolar axon terminals. Thus, these findings may be the characteristics of the calbindin-immunoreactive ON cone bipolar ribbon synapse in sublamina a and can be used to classify the synapse in the retinal circuit research.